Green fluorescent protein (GFP) has had a huge impact on molecular and cell biology because the fluorescent chromophore is formed from a tripeptide sequence within the protein without the involvement of any cofactors other than molecular oxygen. Consequently, GFP can be introduced into live cells or organisms as a fusion protein construct in order to follow localization, processing and interactions of the fusion protein partner. Our interests in these fluorescent proteins are two fold. Firstly, we have a long-standing interest in dissecting the intimate details of structure-function relationships in biological systems. GFP is an intrinsically fascinating system that presents us with the opportunity to investigate how the protein matrix controls the structure and properties of an embedded molecule. Secondly, a deeper understanding of the mechanism of chromophore formation and of how the protein modulates the structure and spectroscopic properties of the chromophore are fundamental to guiding the development of fluorescent proteins with tailored properties and for interpreting data from current applications. There is also a strong drive towards the development of new light sensitive GFPs for studying dynamic processes inside living cells. The current proposal is centered on two hypotheses (i) that changes in light emission from the fluorescent proteins that occur under constant illumination result from light-induced changes in the chromophore structure that are controlled by the protein environment and (ii) that the folded fluorescent protein directs and controls the formation of the chromophore. Based on these hypotheses we will (i) determine the changes in chromophore structure following light absorption and (ii) determine the mechanism of chromophore formation. We will use Raman spectroscopy coupled with other biophysical techniques, such as absorption and fluorescence spectroscopy, to elucidate the changes in chromophore structure that accompany light absorption. In addition, native chemical ligation will be used to insert unnatural amino acids into the chromophore in order to directly probe the mechanism of chromophore formation.